DAG3 Pilot (200 samples), QC report part II - bioinformatics QC vs Lab QC



Identification of ‘problematic’ samples

How to define a ‘bad’ (metagenomic) sequencing run?


  • Low DNA concentration?
  • DNA contamination?
  • ‘Bad’ result of diagnostic gel run?
  • Low number of reads?
  • High level of read duplication?
  • Unusually low or high GC content?
  • Low microbiome diversity?
  • …

For purposes of this analysis, ‘problematic’ samples were defined as:

  • samples with high sequence duplication (>=30% of total reads are duplicate)
  • and/or GC content considerably different from rest of samples (<= 47% or >= 57.5%)


sample tot.seq pct.gc pct.dup UMCG.C_ng_ul novo.C_ng_ul novo.Conclusion2 tot.seq.corr
7020000655314 13607727 57 42.3 41.6 9.6 Regular prep possible 7857782
7020000717918 14165712 46 40.9 32.0 7.4 resend recommended 8366270
7020000774217 24707796 46 27.9 58.1 4.0 resend recommended 17821733
7020000854013 13689929 48 32.6 34.8 6.6 require low DNA library prep 9229750
7020000866417 17151076 45 27.9 118.7 26.2 Regular prep possible 12373644
7020000871032 16918081 54 31.8 67.3 21.0 Regular prep possible 11530518
7020000876431 14700833 46 32.1 39.2 6.4 resend recommended 9985541
7020001036428 12699984 40 61.0 73.2 15.5 Regular prep possible 4953629
7020001093925 14347902 59 16.1 43.3 4.3 resend recommended 12034303
7020001128136 17080328 59 31.2 36.4 8.3 require low DNA library prep 11758098

LAB QC (Novogene)



LAB QC (Novogene)



LAB QC (UMCG)

Our DNA concentration


LAB QC (UMCG)

Batch effects testing (lab parameters):


UMCG: Box 24 seems to be problematic (all samples were labelled as Failed or Hold by Novogene)

  • Box 24: extraction volume was 200 ul, rest of boxes were done with 100 ul
  • different protocol?

Batch effects testing (FastQC results):



Batch effects testing (Taxonomy results):


UMCG QC (Nanodrop) vs Novogene QC (Qubit2):

  • comparison of measurements at UMCG to Novogene report


Post-sequencing QC (FastQC)



FastQC parameters for Novogene QC groups:



FastQC parameters VS DNA concentration (UMCG Nanodrop)



FastQC parameters VS DNA concentration (Novogene Qubit2)



FastQC plots (different FastQC parameters)



Taxonomy



Taxonomy: Taxonomy (Species & Diversity) VS FastQC measurements



Taxonomy: Taxonomy (Species & Diversity) VS Lab measurements



260/280 measurements